- Top of page
- Materials and Methods
- Supporting Information
Photosynthesis and respiration in the plant cell are intimately linked processes; they jointly participate in the exchange of carbon dioxide and oxygen and, as a result, chloroplasts and mitochondria rely on each other for the provision of sugars and ATP, respectively (Nunes-Nesi et al., 2011). Additionally, photorespiratory oxygenation of Ribulose-5-P in chloroplasts leads to enhanced mitochondrial respiration to regenerate substrates for the Calvin cycle (Maurino & Peterhansel, 2010). It has been estimated that reduction of the net photosynthetic CO2 assimilation rate as a result of photorespiration at high temperature in C3 plants like rice can be as high as 25–35% (Sage, 2001). The subtraction of the respiration rate from the photosynthetic rate determines the biomass production in plants and is an important agricultural index for crop production (Amthor, 1989). Efforts to engineer C4 photosynthesis or bypass the role of mitochondria in Ribulose-5-P regeneration in order to eliminate photorespiration in C3 plants are continuing in several cereal crops (Long et al., 2006; Hibberd et al., 2008). However, despite the extensive literature on the differentiation of the plastid from an etioplast to a chloroplast in response to light (Kleffmann et al., 2007; Schattat et al., 2012), and an in-depth understanding of the processes that define the photorespiratory pathway in C3 plants (von Caemmerer & Evans, 2010; Maurino & Peterhansel, 2010), insights are still lacking into the differentiation of mitochondria during the development of photosynthetic cells of plants and into how conserved these processes are between the major C3 dicot crop species and the C3 Poaceae.
In green plant tissues, mitochondria have long been known to undertake different metabolism during dark and light periods. In the dark and at night, organic acids from breakdown of carbohydrates or proteins (Journet et al., 1986; Brouquisse et al., 1998; Hoefnagel et al., 1998) are major substrates for leaf respiration. In the day or in the light, the rate of tricarboxylic acid (TCA) cycle-linked mitochondrial respiration is lowered (Kromer et al., 1993; Atkin et al., 1998; Griffin et al., 2001; Tcherkez et al., 2005, 2008) and oxidation of the amino acid glycine, generated by peroxisomes from chloroplast-derived photorespiratory products, is the major substrate for mitochondrial respiration in C3 plants (Arron & Edwards, 1980; Walker & Oliver, 1986; Lernmark et al., 1990; Padmasree et al., 2002). While this switch between day and night or light and dark function is linked to substrate availability, it is also regulated at the transcriptional and post-translational levels. The expression of genes encoding mitochondrial glycine decarboxylase (GDC) subunits and serine hydroxymethyltransferase (SHMT) in pea leaf has been shown to be highly up-regulated by light in classical etiolated shoot greening experiments (Macherel et al., 1990; Turner et al., 1993). This effect is reportedly regulated by phytochrome-mediated transcriptional control of the promoters of photorespiratory components (Turner et al., 1993; Tepperman et al., 2001). Post-translationally, the phosphorylation and inactivation of the mitochondrial pyruvate dehydrogenase complex, PDC, occurs in light (Tovar-Mendez et al., 2003), and an unknown post-translational inactivation of GDC function occurs in darkness (Lee et al., 2010). Mitochondrial proteins associated with photorespiratory glycine oxidation reactions are always found in highest abundance in photosynthetic tissues and are much lower in abundance in etiolated tissues and in nongreen tissues (Sachlstrom & Ericson, 1984; Newton & Walbot, 1985; Remy et al., 1987; Lind et al., 1991; des Francs-Small et al., 1992; Bardel et al., 2002; Lee et al., 2008, 2011).
Most of what is currently known about plant mitochondria responses to light is derived from studies of C3 dicotyledonous plants such as pea, potato, spinach and Arabidopsis. However, C3 Poaceae represent a larger proportion of crops and the international focus on C4 engineering to decrease photorespiration has more recently turned to work on these grasses, most notably wheat and rice (von Caemmerer et al., 2012; Reynolds et al., 2012). Early studies on the wheat mitochondrial proteome response to light showed significant induction of GDC subunits in isolated mitochondria (Rios et al., 1991), but this was before detailed studies of the specific proteins involved could be performed. In rice, our previous analysis of biogenesis and heterogeneity of mitochondrial proteome using available microarray data revealed that the transcripts for genes encoding mitochondrial photorespiration (GDC and SHMT) have selectively higher steady-state levels in leaf tissues (Huang et al., 2009). However, analysis of the 52 genes in rice predicted to encode enzymes involved in eight steps of the photorespiration pathway, including genes encoding mitochondrial GDC and SHMT subunits, revealed that only one or two genes for each step had higher absolute expression than that of other homolog genes in the same step in response to light, suggesting very selective expression of particular photorespiratory genes in the light in rice (Jung et al., 2008). It was still largely unknown whether these transcriptional changes lead to changes in the abundance of mitochondrial photorespiratory proteins, photorespiration rate or other changes in mitochondrial function in rice. Previous proteomics studies on the response of etiolated rice leaves to light have failed to identify changes in mitochondrial enzymes as a result of the low relative abundance of mitochondria in whole leaf tissue for analysis (Komatsu et al., 1999; Yang et al., 2007). The induction of the photorespiratory apparatus in rice leaves in response to greening in the light not only is a model for studying the initiation of photorespiration, but is also physiologically important because of the practice of low light germination, transplantation, and greening of rice seedlings in the field in many rice-growing regions of the world (De Datta, 1981). Even in case of direct sowing, rice germinates in low-light conditions in muddy paddy fields or underwater and then adapts to higher light intensities when the seedling grows tall enough to reach air and full light intensity (De Datta, 1981).
To better understand the light responsiveness of the protein machinery in rice mitochondria, we investigated changes of metabolite profiles and mitochondrial proteome in 10-d-old etiolated rice plants in response to two intensities of white light (low light (LL), 100 μmol m−2 s−1, and high light (HL), 700 μmol m−2 s−1) for 24 h. Our data revealed that changes in mitochondrial metabolic machinery in response to light correlate with decreased respiration rate and lowered free amino acid content of leaves. Mitochondrial machinery for photorespiration was significantly but differentially induced by light which correlated with a higher photorespiratory capacity in LL- than in HL-treated plants, measured as the postillumination burst (PIB) in respiration.
- Top of page
- Materials and Methods
- Supporting Information
We previously analyzed the biogenesis and heterogeneity of rice mitochondrial proteome using available microarray data and revealed that the transcripts for genes encoding rice mitochondrial photorespiration (GDC and SHMT) have selectively higher steady-state levels in leaf tissues (Huang et al., 2009), much like those in various dicot species (Walker & Oliver, 1986; Macherel et al., 1990, 1992; Rios et al., 1991; Turner et al., 1993; Oliver & Raman, 1995; Lee et al., 2010). In this study, we directly investigated changes of mitochondrial proteome and metabolite profiles in 10-d-old etiolated rice plants in response to two intensities of white light (100 and 700 μmol m−2 s−1) for 24 h, representing the low and high range of light intensities experienced in rice fields (De Datta, 1981). Our findings suggest that mitochondrial composition in etiolated shoots respond differently to LL and HL, with some responses more pronounced under HL and others more evident under LL. Interestingly, chloroplast proteome development, Chl content and the initiation of photosynthesis all showed a greater response to LL than to HL. The majority of the variation in the plant mitochondrial proteome in response to light occurred in proteins involved in the mitochondrial photorespiratory machinery, the TCA cycle, amino acid, and branch chain amino acid metabolism. Two notable differences other than this major C/N machinery were the light-dependent increases in HSP70 and NDPKIII. We observed significant changes in metabolite concentrations in response to light, dominated by the general decrease in amino acid content and increase of organic acid content.
NDPKIII in rice
Mitochondrial NDPK was increased under both LL and HL conditions (Table 3). In Arabidopsis, mitochondrial NDPKIII is reported to be dual-targeted to chloroplast based on green fluorescent protein localization and western bolt experiments (Hammargren et al., 2007). In rice, NDPKIII is exclusively mitochondrial-targeted, while NDPKII is targeted to plastids (Kihara et al., 2011). Enzymatically NDPK is involved in nucleotide conversion (classically ATP + guanosine diphosphate (GDP) ADP + guanosine triphosphate (GTP)), and a role for mitochondrial NDPK in phosphor transfer within nucleotide pools is often discussed (Johansson et al., 2004). However, NDPKs are more widely involved in signaling processes in a number of species, notably in light signaling, through phytochrome-mediated processes in plants (Im et al., 2004) or via singlet oxygen processes in fungi such as Neurospora crassa (Yoshida & Hasunuma, 2006). In these roles, NDPK directly converts bound GDP to GTP while the nucleotides are still bound to G proteins (Randazzo et al., 1991). Knowledge of NDPK binding partners in plant mitochondria is very limited; the only report is NDPK interaction with a 86 kDa heat shock protein in pea during heat stress (Galvis et al., 2001). In mammals, succinyl-CoA-ligase is a binding partner for NDPK in mitochondria (Kowluru et al., 2002) and two mitochondrial S-CoA-L proteins (Os07g38970, Os02g40830) were decreased in abundance in response to light in rice (Table 3). However, if NDPKIII in rice is in the intermembrane space as it is in Arabidopsis, pea and yeast (but not in mammals), then NDPK and S-CoA-L proteins will not be able to interact in vivo.
TCA cycle, amino acid and organic acid metabolism
We observed an apparent reduction of TCA cycle metabolism in response to light treatments, as supported by accumulation of TCA cycle substrates and reduction in abundance of TCA cycle-related enzymes, especially under HL treatment (Figs 2, 3). These results were consistent with previous studies showing that the rate of TCA cycle-linked mitochondrial respiration in the light is lower than in darkness (Kromer et al., 1993; Atkin et al., 1998; Griffin et al., 2001; Tcherkez et al., 2005, 2008). Our results support the proposition by Tcherkez et al. (2008) in leaves of Xanthium strumarium that the decrease focuses on the early and middle steps of the TCA cycle. In Arabidopsis shoots, a similar decrease in the abundance of enzymes in the early and middle steps of the TCA cycle occurred by analysis of diurnal changes in mitochondrial function (Lee et al., 2010). Protein abundance of mitochondrial enzymes involved in amino acid and branch chain metabolism in rice were also decreased by light (Table 3), which was in general agreement with diurnal changes in Arabidopsis mitochondrial amino acid metabolism (Lee et al., 2010). Given the sophisticated pathways of amino acid synthesis and degradation in plant cells, such a reduction in mitochondrial amino acid metabolism could contribute to the changes of amino acid abundances observed in the light (Fig. 3). The development of chloroplasts from etiolated plastids requires a considerable investment of amino acids for protein synthesis during the transition to the light. Such development is delayed by HL treatment compared with LL treatment, as indicated by Chl content, and correlates to the larger residual amino acid content in HL-treated shoots than in LL-treated shoots (Table 1, Fig. 3). Our analysis of Chl content in plants subjected to different light intensities is also consistent with the report that higher light treatment can lower Chl content, even though maximal growth occurs in higher light regimes (Fu et al., 2011), and that shade species maintain higher Chl content in order to maximize light-use efficiency (Boardman, 1977).
Photorespiration in rice
Glycine is one of relatively few amino acids in etiolated rice shoot that was not decreased in abundance by light (Fig. 3). Mitochondrial proteins involved in glycine metabolism, such as GDC subunits and SHMT, were significantly increased in abundance (Table 3, Fig. 3). The induction of glycine-dependent photorespiration proteins by light is widely reported in other plant species, such as Arabidopsis, pea and wheat (Rios et al., 1991; Rogers et al., 1991; Lee et al., 2010). However, the abundance of GDC-H proteins under both light conditions studied here were unchanged (Table 3, Fig. 2), which is in contrast to the common observation of light-induced GDC H proteins in other plant species (Rios et al., 1991). The transcript for GDC-H (Os10g31780) is also reported to respond to light in rice (Jung et al., 2008), so it is possible that this GDC-H protein might be regulated at the post-transcriptional/-translational level or via protein degradation. Previously, light was reported to induce similar gene expression patterns of GDC subunits and photosynthetic genes such as Rubisco small subunit RbsS, but GDC translation was clearly delayed compared with Rubisco (Oliver & Raman, 1995; Vauclare et al., 1996). GDC is also known to be susceptible to oxidative damage by ROS in mitochondria (Douce et al., 2001) and GDC-H protein lipoic acid is targeted by lipid peroxides, which could make GDC-H a target for degradation in mitochondria (Taylor et al., 2002). Under sudden HL treatment, rice mitochondria may be under enhanced light stress, as indicated by induction of heat shock protein 70 (Os09g31486). GDC-H could thus be turned over rapidly after its synthesis. Interestingly, the GDC-L subunit (Os01g22520), which is known to contain cysteine residues sensitive to damage, was reduced in abundance under HL (Table 3). The decrease in the GDC-L subunit under HL might explain, at least in part, why there is no apparent photorespiration rate in these HL-exposed shoots. Unlike the pyruvate dehydrogenase complex and the 2-oxoglutarate dehydrogenase complex of the TCA cycle that form large complexes with strict stoichiometry (Mooney et al., 2002), GDC forms transient interactions across the network of subunits to facilitate the oxidation of glycine to serine (Douce et al., 2001). The changes in GDC stoichiometry observed here, coupled with large differences in photorespiratory rate (Fig. 4), highlight the mitochondrial rate-limiting components contributing to photorespiratory capacity.
In conclusion, our data show that light-induced changes in the etiolated rice shoot led to a shift in its mitochondrial metabolic machinery from the TCA cycle, amino acid-related metabolism to photorespiration-related metabolism. The unexpected lack of light induction of GDC-H and -L subunit proteins in rice shoot mitochondria warrants further attention to understand the mechanism behind this phenomenon and its implications for photorespiratory development in light-exposed rice seedlings. Detailed analyses of light-induced greening of dark-germinated seedlings, kinetic responses to different light intensities between LL and HL, and diurnal changes in the rice leaf mitochondrial metabolism are required to better mimic nature field conditions and provide further insights into how mitochondrial metabolism adapts to its role in photosynthetic rice tissues.